The kinetochore is the chormosomal structure, located at the centromere, to which spindle microtubules attach during mitosis and meiosis. Despite the obvious importance of the kinetochore in the life cycle of the cell, relatively few molecular details of kinetochore structure or function are known. This application proposes a combined biochemical and genetic study of the kinetochore complex of yeast (Saccharomyces cerevisiae). Yeast is an ideal eukaryote for this project, because the precise centromere DNA sequences of yeast are known, and centromere function can be assayed in vivo. Furthermore, the elegant genetic methods available with yeast make detailed gnetic analyses possible. Using specific DNA-protein binding assays, proteins which bind to yeast centromere DNA will be isolated and their precise binding sites determined. Centromere DNA mutated at those binding sites will be tested for its ability to function in vivo. A correlation between binding in vitro and centromere function in vivo will be interpreted to mean that that particular binding interaction is required for kinetochore function, and therefore that the binding protein is essential. Proteins so identified will be purified further and used to produce specific antibodies. The antibodies will be used to screen wavelength gt11 expression libraries and thereby isolate the binding protein genes. Once the genes are cloned and characterized, null and conditional (ts) mutants will be created utilizing gene disruption and replacement techniques. Long-range studies will involve using the centromere binding proteins and their genes to identify interacting genes and gene products, extending the study to other components of the spindle apparatus. Results obtained from the yeast system are likely to be directly applicable to higher eukaryotic systems. In most major respects, mitosis in yeast is similar to mitosis in higher eukaryotes, and at least some proteins of the yeast mitotic apparatus cross-react immunologically with their human counterparts.